Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of par affin blocks stored for different periods of time with P-33-labeled ribopro bes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP- 2), Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage per iod of the sections. For slides hybridized with the MMP-2 riboprobe, differ ences in signal intensity could be detected, albeit inconsistently, with th e phosphorimager, although microscopic examination consistently indicated s tronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepare d sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.